1. Field of the Invention
This invention relates generally to charge coupled device (CCD) cameras and more specifically to CCD cameras used in transmission electron microscopes (TEMs).
2. Description of the Related Art
Slow-scan CCDs are becoming widely used on electron microscopes, and under some conditions they provide an improvement in data quality over photographic film as well as the obvious advantages of direct digital readout. However, the CCD performance is seriously degraded on Intermediate Voltage Electron Microscopes (IVEMs) operating at 300-400 kV compared to the more conventional 100 kV microscopes. Because the IVEM is becoming more widely used, poor performance of the CCD camera is limiting.
Up until now, the performance of the CCD with high energy electrons has been significantly inferior to that of photographic film, making it impractical to use the CCD for image recording in many biological applications. High-quality image data cannot be collected directly with the CCD camera as needed for high-resolution electron crystallography of proteins and tomographic studies of cell ultrastructure.
There are numerous benefits to raising the voltage in an electron microscope. As the accelerating voltage increases, both elastic and inelastic scattering cross sections decrease, so that thicker specimens can be used. This is of particular importance with frozen hydrated preparations of larger molecules and assemblies and with embedded sections studied by tomography for three-dimensional visualization. Also the single-scattering approximations that are fundamental to much of the image interpretation and processing improve with increasing voltage, so that the data is a better representation of the specimen. The envelope function for temporal coherence improves, so it is easier to routinely obtain high resolution. Since the depth of field is larger, the area of a tilted specimen that is close to focus is larger. The diffraction-limited spot size is smaller, so spot-scan imaging can be done with a smaller illumination spot. Focus ramp correction with spot scan imaging can even ensure that the entire image will be at essentially the same focus. The gun brightness is higher, providing some improvement in coherence and/or exposure time. Each of these factors provides an incremental improvement, and taken together the difference is quite substantial. It is indeed this combination of factors that is still driving microscope manufacturers to improve intermediate and high voltage microscopes, and the voltage of what is considered the "routine" microscope continues to increase.
In biological applications, high voltage microscopes were initially used exclusively for their high penetration with thicker specimens. Stable IVEMs, operating at 300-400 kV are used for high resolution protein structure work as well. Camera resolution remains a limiting factor in using current IVEMs. In many potential applications, e.g. crystallography and the study of single molecule structures, digitization and high resolution CCD instrumentation would be highly advantageous to study the structures.
Electron microscopy would be enhanced by improved data collection and processing techniques. Among the advances that could greatly improve the efficiency of data collection is the development of the full potential of CCD cameras to electron microscopy. Slow-scan CCDs are used, and in some instances have completely replaced photographic film for data collection. Particularly with 100 kV microscopes, there are suggestions that the CCD is at least as good as film. However, virtually all of the quantitative analyses of CCD performance demonstrate that the quality of data collected with the CCD is compromised by certain of the system characteristics. This is true at any voltage but the problems increase with increasing voltage. Of particular impact is the point spread function, which is broadened by scattering (lateral straggling) of the electrons within the scintillator. This problem is especially aggravated at higher voltages, which appears to be the one disadvantage of the intermediate voltage microscopes. Given the number of advantages of high voltage, though, it is apparent that the WEM will more and more be the instrument of choice for structural studies.
A review of the literature gives a rather unfavorable picture of the potential for using a CCD on an IVEM. Daberkow et al. "Development and performance of a fast fibre-plate coupled CCD camera at medium energy and image processing system for electron holography" Ultralicroscopy 64, 35-48 (1996) have presented calculations and measurements that show a drastic falloff in the detective quantum efficiency (DQE) on going from 100 to 300 kV. To some extent the DQE loss can be avoided with a thick scintillator, but only at the cost of a rather wide point spread function (PSF); for example the PSF is wider than 60 .mu.m for any condition that provides a DQE of over 0.5 at 300 kV. The situation is not quite as bad at 200 kV, but still substantially worse than 100 kV. Faruqi et al. "A high sensitivity imaging detector for electron microscopy" Nucl. Instrum. & Methods in Phys. Res. A 367, 408-412 (1995) also used a thicker scintillator to improve sensitivity, but obtained a PSF well over 50 .mu.m (FWHM) even at 120 kV. Weickenmeier, et al. "Quantitative characterization of point spread function and detection quantum efficiency for a YAG scintillator slow scan CCD camera" Optik 99, 147-154 (1995) measured a modulation transfer function (MTF) for CCD systems that falls below 0.2 even at 120 kV. DeRuijter "Imaging properties and applications of slow-scan charge-coupled device cameras suitable for electron microscopy" Micron 26, 247-275 (1995) compared 100 and 300 kV operation, showing a loss of about a factor of two in MTF at the higher voltage, and also provides a good discussion of other noise sources that have made some MTF measurements appear anomalously favorable. The main problem is that noise within the scintillator, such as from the wide pulse height distribution of photons per incident electron, is not damped by the MTF and thus adds a finite amplitude to the power spectrum at high spatial frequencies. MTF measurements based only on the Fourier transform or autocorrelation of the input will be biased toward high values by this noise source. Van Zwet and Zandbergen "Measurement of the modulation transfer function of a slow-scan CCD camera on a TEM using a thin amorphous film as test signal" Ultramicroscopy 64, 49-55 (1996) illustrate the difference in MTF measurements made with different techniques. Results presented by Sherman et al. "Performance of a slow-scan CCD camera for macromolecular imaging in a 400 kV electron cryomicroscope" Micron 27, 129-139 (1996) are among the most favorable, but still show a strong loss of both signal and signal to noise ratio at high resolution, compared to film.